The broad objective of this proposal is to continue development of hairpin ribozyme as an agent for the efficient specific cleavage of heterologous RNA sequences. By developing and engineering this ribozyme to efficiently cleave specific sequences in the human immunodeficiency virus type 1 (HIV-1), it has potential application as an AIDS therapeutic. The first specific aim of this proposal is to develop optimal hairpin ribozymes for specific sites in the HIV-1 sequence. Initially, the ribozymes to be developed are designed to cleave specific sites in the gag, tat and pol transcripts of HIV-1. Both the conventional hairpin ribozyme and a newly engineered hairpin tetraloop ribozyme will be used. The second aim is to test these ribozymes in vivo (mammalian cell culture) for their ability to cleave HIV-1 sequences. Both phenotypic and molecular (RNA) analyses will be done in both systems to determine in vivo effectiveness. The third aim is to test in vivo activity of a newly developed and catalytically improved hairpin tetraloop ribozyme designed to cleave a target sequence in the HGPRT system. The HGPRT system is an excellent model and will be used for the initial determination of in vivo activity of this hairpin tetraloop ribozyme. Both phenotypic (resistance to 8-azaquanine) and molecular (RNA) effects will be determined. Results to date suggest that the hairpin ribozyme may be an important addition to the therapeutic modalities available for the treatment of AIDS. Hairpin based ribozymes may be particularly advantageous since the conditions required for their optimum function are near physiological, and they are capable of very high catalytic efficiencies. In addition to HIV-1, this approach should also be applicable to eliminate a wide variety of deleterious RNAs whether cellular or viral. The significance of which is far reaching.